Effects of sporoderm-broken germination activated ganoderma spores on promotion of proliferation and/or differentiation of neural stem cells in injured spinal cord

ABSTRACT

The present invention provides a method for promoting proliferation and/or differentiation of neural stem cells in a mammal, which comprises orally administering sporoderm-broken germination activated  Ganoderma lucidum  spores (GASP) to the mammal. The effects of GASP on proliferation and/or differentiation of neural stem cells are particularly prominent in mammals after a spinal cord injury.

RELATED INVENTION

The present invention is a Continuation-In-Part (CIP) Application ofU.S. patent application Ser. No. 10/631,809, filed on Aug. 1, 2003,which is herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a method for promoting proliferationand/or differentiation of neural stem cells in a mammal, particularlyafter the mammal has a spinal cord injury, by administering to themammal sporoderm-broken germination activated ganoderma spores (GASP).

BACKGROUND OF THE INVENTION

The spinal cord coordinates the body's movement and sensation to andfrom the brain. It is a complex organ containing nerve cells (also knownas neurons), and supporting cells (also known as neuroglial cells orglial cells). A typical neuron consists of a cell body, containing thenucleus and the surrounding cytoplasm (perikaryon); several shortradiating processes (dendrites); and one long process (the axon), whichterminates in twiglike branches (telodendrons) and may have branches(collaterals) projecting along its course. The axon together with itscovering or sheath forms the nerve fiber. The neuroglial or glial cellsform the supporting structure of nervous tissue. There are three typesof glial cells, which are astrocytes, oligodendrocytes, and microglia.Astrocytes and oligodendrocytes (collectively macroglia) are ofectodermal origin. These cells far outnumber neurons in the brain andspinal cord and perform many essential functions. The oligodendrocytecreates the myelin sheaths that insulate axons and improve the speed andreliability of nerve signal transmission. Astrocytes, large star-shapedglial cells, regulate the composition of the fluids that surroundneurons. Some of these cells also form scar tissue after injury.Microglia are of mesodermal origin. They are smaller cells that becomeactivated in response to injury and help clean up waste products. All ofthese glial cells produce substances that support neuron survival andinfluence axon growth.

Many axons in the spinal cord are covered by sheaths of an insulatingsubstance called myelin, which gives them a whitish appearance;therefore, the region in which they lie is called “white matter.” Theneurons themselves, with their tree-like dendrites that receive signalsfrom other neurons, make up “gray matter.” This gray matter lies in abutterfly-shaped region in the center of the spinal cord. Like thebrain, the spinal cord is enclosed in three membranes (meninges): thepia mater, the arachnoid, and the dura mater. The spinal cord is thensurrounded by rings of bone called vertebra.

The spinal cord and the brain together make up the central nervoussystem (CNS). Unlike neurons of the peripheral nervous system (PNS),which carry signals to the limbs, torso, and other parts of the body, itwas generally believe that new neurons could not be generated in theadult mammalian brain and neurons of the CNS do not regenerate afterinjury. More recently, findings that neurons can be renewed in certainregions of the adult CNS, e.g., in the olfactory bulb, where signalsfrom neurons from the organ of smell reach the brain and in the dentategyrus of hippocampus, have been reported. Since neurons are unable todivide, the addition of new neurons suggested the existence of immaturecells, i.e., progenitor or stem cells, which may generate neurons.

Stem cells are undifferentiated cells capable of proliferation,self-maintenance, and production of a large number of differentiated,functional progeny, regenerating tissue after injury. Stem cellsisolated from the early embryonic blastula, i.e., prior to gastrulation,can produce cell types of all different lineages (Keller, Curr. Opin.Cell Biol., 7:862-869 (1995)). Stem cells in adults, however, play therole of replacing cells which have been lost by natural cell death,injury or disease. When transplanted into the brain, these neural stemcells survived and differentiated into neurons and neuroglial cells.(Johansson et al., Cell (1999), 96(1): 25-34). There are also certainnumbers of neural stem cells in the spinal cord. They are proliferatingcells in the ependyma of the spinal central canal in adult mammals.(Frisen et al., J. Cell Biol. (1995), 131:453-464; Yamamoto et al., Exp.Neurol. (2001), 172:115-127)). A method for isolation of ependymalneural stem cells have recently been described in U.S. Pat. No.6,541,247.

The existence of neural stem cells in the adult mammalian CNS was firstdemonstrated by culturing cells from the adult rat brain and spinalcord. Under certain culture conditions, a population of multipotentneural stem cells can be propagated. (Reynolds et al., Science (1992)255:1707-1710). Under these conditions, single cells proliferate invitro and the progeny forms a cluster of aggregated cells. Such cellclones detach from the culture dish after a few days in vitro. The cellscontinue to proliferate and form a characteristic spheroid cellaggregate, referred to as a neurosphere, of tightly clustered cells, allof which are derived from a single cell. Most of the cells in theneurosphere express nestin, an intermediate filament found inneuroepithelial stem cells, (Lendahl et al., Cell (1990), 60:585-595),but not markers typical for differentiated cells. These undifferentiatedcells rapidly differentiate if plated on an adhesive substrate or ifserum is added to the culture medium. Importantly, a clone of cellsderived from a single cell can generate neurons, astrocytes andoligodendrocytes, demonstrating that at least the initial cell wasmultipotent (Reynolds et al., Science, ibid.). Moreover, if a cell cloneis dissociated, many of the cells will form new clusters ofundifferentiated multipotent cells, thus fulfilling the criteria forbeing stem cells.

The development of the mammalian CNS begins in the early stage of fetaldevelopment and continues until the post-natal period. (U.S. Pat. No.6,497,872). The first step in neural development is cell birth, which isthe precise temporal and spatial sequence in which stem cells and stemcell progeny (i.e., daughter stem cells and progenitor cells)proliferate. Proliferating cells will give rise to neuroblasts,glioblasts and new stem cells.

The second step is a period of cell type differentiation and migrationwhen undifferentiated progenitor cells differentiate into neuroblastsand gliolblasts which give rise to neurons and glial cells and migrateto their final positions. Cells which are derived from the neural tubegive rise to neurons and glia of the CNS, while cells derived from theneural crest give rise to the cells of the peripheral nervous system(PNS). Certain factors present during development, such as nerve growthfactor (NGF), promote the growth of neural cells. NGF is secreted bycells of the neural crest and stimulates the sprouting and growth of theneuronal axons.

The third step in development occurs when cells acquire specificphenotypic qualities, such as the expression of particularneurotransmitters. At this time, neurons also extend processes whichsynapse on their targets. Neurons are generated primarily during thefetal period, while oligodendrocytes and astrocytes are generated duringthe early post-natal period. By the late post-natal period, the CNS hasits full complement of nerve cells.

The final step of CNS development is selective cell death, wherein thedegeneration and death of specific cells, fibers and synapticconnections “fine-tune” the complex circuitry of the nervous system.This “fine-tuning” continues throughout the life of the host. Later inlife, selective degeneration due to aging, infection and other unknownetiologies can lead to neurodegenerative diseases.

There is increasing evidence that nervous system injuries may affectstem cells in the adult CNS. After both spinal cord and brain injuries,nestin expression is increased in cells lining the central canal and inthe subventricular zone, respectively. (Frisen et al., J. Cell Biol.,Ibid.). These cells may be derived from stem cells. With time, nestinexpressing cells are seen progressively further from the central canaland the lateral ventricle and these cells express astrocytic markers.(Frisen et al., J. Cell Biol., ibid.). These data demonstrate that stemcells or progenitor cells residing by the ventricular system are inducedto proliferate, migrate toward the site of the injury and differentiateto astrocytes.

Currently, no methods are available in clinical practice to stimulategeneration of new cells in the nervous system. Transplantation of cellsfrom human embryos or animals have been tested clinically with someencouraging results. However, these methods have several problems,mainly ethical and immunological, which makes it very unlikely that theywill be used in any larger number of patients. Accordingly, thediscovery of a proliferation and differentiation factor on neural stemcells in the adult CNS of mammals is important and may make it possibleto develop strategies to stimulate generation of new neurons or glialcells.

Recently, Cheung et al., FEBS Lett. (2000), 486: 291-296, disclose thatGanoderma lucidum extract induces neuronal differentiation in vitro,using a primary neuronal cell system (i.e., PC12 cells). The PC12 cellsare derived from rat pheochromocytoma cells which respond to the nervegrowth factor (NGF). The Ganoderma lucidum extract described by Cheunget al. is made from the fruit bodies of the Ganoderma lucidum.

In the invention to be presented in the following sections, a novel useof sporoderm-broken germination-activated Ganoderma spores (GASP) fromGanoderma lucidum as an effective, safe and practical alternative toinduce the proliferation of self neural stem cells of the injured CNSand migration to the injured area and further differentiation intoneurons for replacement of damaged or lost neurons is described. In theparent application of the instant invention, we have described the noveluse of GASP in promoting the motor neuron survival and axon regenerationand its use in treatment of related spinal cord injury. The GASP havealso previously been disclosed for use in treating patients with cancer,AIDS, hepatitis, diabetes, and cardiovascular diseases, and can preventor inhibit free radical oxidation and hepatotoxic effects. See U.S. Pat.Nos. 6,316,002 and 6,468,542, which are incorporated herein byreference. A further benefit of using the GASP is that they arenon-toxic so that higher dosage can be prescribed to the patients.

SUMMARY OF THE INVENTION

The present invention provides a method for promoting proliferationand/or differentiation of neural stem cells in a mammal, byadministering an effective amount of sporoderm-broken, germinationactivated Ganoderma spores (GASP) into a mammal, preferably human.Gandoderm (Ganoderma lucidum Leyss ex Fr. Karst) is a polyporous fungus.It belongs to the class of Basidiomycetes, the family of Polypolaceae,and the genus of Ganoderma. The GASP is especially effective onpromoting proliferation and/or differentiation of neural stem cell afterthe mammal has a spinal cord injury. The causes for a spinal cord injuryinclude, but are not limited to, compression or severance of the spinalcord, trauma (such as car accident, violence, falls, sports etc.), or adisease (such as polio, spina bifida, or Friedreich's Ataxia). Inaddition, the spinal cord injury can be due to damage or death ofneurons within the injured spinal cord or crush of axons within theinjured spinal cord. The GASP are preferred to be orally administered toa mammal, including a human, in the amount of about 0.5-15 g per kg ofbody weight per day, most favorably about 8 g per kg of body weight perday.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the BrdU fluorescent immunohistochemistry of across-section of a rat normal spinal cord. Arrow (↑) indicates theBrdU-positive cells in the ependyma of the spinal central canal underfluorescent microscope at 200 fold magnification. Ependyma is the liningmembrane of the ventricles of the brain and of the central canal of thespinal cord.

FIG. 2 shows the BrdU fluorescent immunohistochemistry of across-section of a rat spinal cord 1 day after a spinal cord injury.Arrow (↑) indicates the BrdU-positive cells in the ependyma of thespinal central canal and the surrounding areas under fluorescentmicroscope at 200 fold magnification.

FIG. 3 shows the nestin fluorescent immunohistochemistry of across-section of a rat spinal cord 1 day after a spinal cord injury.Arrow (↑) indicates nestin-positive cells in the ependyma of the spinalcentral canal and the surrounding areas under fluorescent microscope at200 fold magnification.

FIG. 4 shows the nestin fluorescent immunohistochemistry of across-section of a rat spinal cord 4 weeks after a spinal cord injury.Arrow (↑) indicates the nestin-positive cells in the injured whitematter of the injured/treatment group under fluorescent microscope at200 fold magnification.

FIG. 5A shows the dual BrdU and nestin fluorescent immunohistochemistryof a cross-section of injured spinal cord at 4 weeks after spinalinjury. Arrow (↑) indicates the BrdU-positive cells in the white matterof the injured spinal cord of the injured/treatment group underfluorescent microscope at 200 fold magnification.

FIG. 5B shows the same view as in FIG. 5A. Arrow (↑) indicates that someof the BrdU-positive cells also expressed nestin under fluorescentmicroscope at 200 fold magnification.

FIG. 6A shows a dual BrdU and NF fluorescent immunohistochemistry of across-section of injured spinal cord at 4 weeks after spinal injury.Arrow (↑) indicates the BrdU-positive cells in the spinal white matterof the injured/treatment group under fluorescent microscope at 200 foldmagnification.

FIG. 6B shows the same view as in FIG. 6A. Arrow (↑) indicates some ofthe BrdU-positive cells also expressed NF under fluorescent microscopeat 200 fold magnification.

FIG. 7A shows the dual BrdU and oligodendrocytin fluorescentimmunohistochemistry of a cross-section of injured spinal cord at 4weeks after spinal injury. Arrow (↑) indicates the BrdU-positive cellsin the spinal white matter of the injured/treatment group underfluorescent microscope at 200 fold magnification.

FIG. 7B shows the same view as in FIG. 7A. Arrow (↑) indicates some ofthe BrdU-positive cells also expressed oligodendrocytin underfluorescent microscope at 200 fold magnification.

FIG. 8A shows the dual BrdU and GFAP fluorescent immunohistochemistry ofa cross-section of injured spinal cord at 4 weeks after spinal injury.Arrow (↑) indicates the BrdU-positive cells in the spinal white matterof the injured/treatment group under fluorescent microscope at 200 foldmagnification.

FIG. 8B shows the same view as in FIG. 8A. Arrow (↑) indicates some ofthe BrdU-positive cells also expressed GFAP under fluorescent microscopeat 200 fold magnification.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for promoting the proliferationand/or differentiation of neural stem cells in a mammal, particularlyafter a spinal cord injury. The method involves administering aneffective amount of germination activated Ganoderma spore powder (GASP)into a mammal having spinal cord injury.

Spinal cord injury has been a serious problem in humans, which, in manycase, leads to complete or incomplete loss of motion or sensoryfunction. The seriousness of spinal cord injury is primarily due to thedifficulty of recovery from injury because the neurons in the spinalcord are very specialized and unable to divide and create new cells. Inthe United States, approximately 450,000 people live with spinal cordinjury, and there are about 10,000 new cases every year, mostlyinvolving males between ages of 16 to 30. Although spinal cord is wellprotected, spinal cord injury does occur due to causes such as trauma(e.g., car accident, violence, falls, sports) and disease (e.g., polio,spina bifida, Friedreich's Ataxia).

Ganoderma (Ganoderma lucidum Leyss ex Fr. Karst) is a polyporous funguswhich belongs to the class Basidiomycetes, the family Polypolaceae, andthe genus Ganoderma. Ganoderma spores are tiny and mist-like spores of5˜8 μm in sizes which have extremely hard and resilient, double-layerepispores, thus making them difficult to break open. The spores containhigh concentrations of many bioactive substances, including, but are notlimited to, polyunsaturated fatty acids, polysaccharides, vitamins,sterols, trace minerals, amino acids, and triterpenes. The GASP used inthe present invention are sporoderm-broken (i.e., the double-layerepispores of the spores are broken so that the bioactive substanceswithin the spores are released), which is produced by the methoddescribed in U.S. Pat. No. 6,316,002 (“the '002 patent). The entirecontent of the '002 patent is herein incorporated by reference. Throughthe unique spore-breaking method described in the '002 patent, thebioactive substances within the GASP are recovered in high yields andthe functional activities of the bioactive substances are successfullypreserved.

As shown below is a general description of the method used in the '002patent, which leads to the production of the GASP:

I. Soaking to induce germination: Mature and perfect spores of Ganodermalucidum were carefully selected to carry out a soaking process to inducegermination. Spores were kept in clear or distilled water, biologicalsaline solution, or other nutritional solutions that could enable thespores of Ganoderma lucidum to germinate rapidly. Examples ofnutritional solutions include coconut juice or a 1-5% malt extractsolution, 0.5-25% extracts of Ganoderma lucidum sporocarps or Ganodermalucidum capillitia, 0.1-5% of culture solution containing biotin, 0.1-3%of culture solution containing monobasic potassium phosphate andmagnesium sulfate. The choice of solution would depend on the soakingtime required, the amount of spores to be processed and other suchfactors as availability of materials. One or more of the abovegermination solutions could be used, with the amount added being 0.1-5times the weight of the spores of Ganoderma lucidum. The soaking timecan be determined according to the temperature of the water, and usuallythe soaking was carried out for 30 min to 8 h with the temperature ofthe water at 20-43° C. Preferably soaking times were 2-4 hours, andtemperature of the water was 25-35° C.

II. Activation culture: The spores of Ganoderma lucidum were removedfrom the soaking solution and excess water was eliminated by allowing itto drip. The spores were then placed in a well-ventilated culturing boxat a constant temperature and humidity so that spore activation culturecould be carried out. The relative humidity of the culture was generallyset at 65-98%, the culture temperature at 18-48° C. and the activationtime lasted from 30 min to 24 h. Preferably humidity is 85-97% andtemperature is 25-35° C. Using the method provided by the presentinvention, the activation of spores of Ganoderma lucidum reached a rateof more than 95%. During activation, the cell walls of the spores of redGanoderma lucidum were clearly softened such that it was easier topenetrate the cell walls of the spores.

III. Treatment of the epispores: After the germination activationprocess, the spores were treated by enzymolysis. This process wascarried out at a low temperature and under conditions such that enzymeactivity was maintained, using chitinase, cellulase, or other enzymes,which are commonly used in the industry. The process was complete whenthe epispores lost their resilience and became brittle. Alternatively,physical treatments were carried out to penetrate the cell walls, forexample, micronization, roll pressing, grinding, super high pressuremicrostream treatment, and other mechanical methods commonly used in theindustry could be carried out, with a penetration rate of over 99%.

IV. Drying or extraction: Drying was carried out at low temperatureusing standard methods including freeze-drying or vacuum-drying etc.,which are commonly used in the industry. The obtained product had amoisture content less than 4%. After drying, the bioactive substanceswere extracted by water or alcohol, or by thin film condensation. Theextracted bioactive substances could be further purified by dialysis toensure no contamination in the final products.

V. Pharmaceutical formulations of the bioactive substances: Thebioactive substances can then be made into purified powders, extractpastes, solutions for injection, or for oral consumption. The inventionalso encompasses the manufacture of pharmaceutical preparations of theactive substances, using well-known expedients and methods ofmanufacture known in the art. In addition, the bioactive substances canbe dosed by any convenient method including tablets, capsules,solutions, suppositories, nasal sprays, paranterals, or injectiondevices. The choice of method for administration is determined by theclinical situation of the patient. The bioactive substances of thepresent invention, produced by the methods described, include activegenes, inducers of the biotic potential promotor, inducers of themulticellular activator, inducers of interferon, lactone A, ganodermapolysaccharide, ganoderma spore fatty acids, ganoderma spore long chainalkyl hydrocarbon, ganoderma triterpenes, sterols, superoxide dismutase,vitamin E, active glycoprotein, certain growth factors, ganoderma acidA, superoxide dismutases (SOD), active glycoproteins, multiple activeenzymes, and growth factors and so on. These bioactive substances, in awhole, contribute to the therapeutic uses described in the latersections.

GASP are non-toxic. The preferred method for administering GASP isthrough oral uptake. Currently, GASP are approved by the Food and DrugAdministration (FDA) to be used as dietary supplement in the capsuleform under the name of Enhanvol® and Holistol, sold by Enhan TechnologyHoldings International Co., Ltd. in Hong Kong. Each capsule of GASPcontains 0.3 g of GASP. The recommended dosage of GASP, when used asdietary supplement, is 4 times every day, 4 capsules each time. Thus,for an adult of 60 kg, the daily dosage of GASP as dietary supplement isat about 0.08 g/kg of body weight per day.

It has been shown, however, that no physiological and pathologicalabnormalities were found when 8 g/kg/day of GASP were given to patientsand animals. 0.5 g to 15 g/kg/day of GASP have been given to animals anddemonstrated significant effects on treatment of spinal cord injury.However, it is understood that the dosage for any particular patientdepends upon a variety of factors, including age, body weight, generalhealth, sex, diet, time of administration, route of administration, rateof excretion, drug combination and the severity of the disease. Forthese reasons, dosing is left to the discretion of the skilledclinician.

The present invention is conducted using a rat model. According to Metzet al., J Neurotrauma (2000), 17(1):1-17), which is herein incorporatedby reference, the functional, electrophysiological and morphologicaloutcome parameters following spinal cord injury in rats can beextrapolated for those in humans. Metz et al. collected data from humanpatients with chronic spinal cord injury and compared them to those ofrats with contusion spinal cord injury induced by a weight-drop. Theresults suggest an analogous relationship in rats and humans withrespect to functional, electrophysiological, and morphological outcomes,which demonstrates that rat can serve as an adequate animal model forresearch on functional and morphological changes after spinal cordinjury and the effects of new treatment strategies.

In the present invention, five (5) fluorescent neruonal markers wereused, which are BrdU, nestin, neurofilament (NF), oligodendrocytin, andglial fibrillary acidic protein (GFAP), to explore the proliferation ofcells in general (based on the staining of BrdU), the undifferentiatedneural stem cell (based on the staining of nestin), the newly generatedneurons from neural stem cell (based on the staining of NF), the newlygenerated oligodendrocytes from neural stem cell (based on the stainingof oligodendrocytin), and the newly generated astrocytes (based on thestaining of GFAP).

The following experimental designs are illustrative, but not limitingthe scope of the present invention. Reasonable variations, such as thoseoccur to reasonable artisan, can be made herein without departing fromthe scope of the present invention.

Experiment 1 Preparation of Spinal Hemisection Animal Model, Injectionof BrdU, and Treatment of GASP

Animal grouping. Twenty-six (26) female SD rats (150-180 g) werepurchased from the Experimental Animal Center of the Sun Yat-senUniversity. The animals were separated into 3 groups: the normal controlgroup (2 rats), the injured/control group (12 rats: 2 rats in the 1-daygroup, 5 rats each in the 2-week and 4-week groups) and theinjured/treatment group (12 rats: 2 rats in the 1-day group, 5 rats eachin the 2-week and 4-week groups).

Animal Model preparation. 1% Pentobarbital sodium (35˜40 mg/kg) wasinjected intraperitoneally to anesthetize the rats in theinjured/control group and the injured/treatment group. The skin on theback of the rat was cut open at the centerline. The thoracic (T) 12spinal segment was exposed under a surgical microscope and hemisectionof the right side of the spinal cord was performed. After surgery,penicillin was injected intramuscularly.

BrdU injection and treatment: The animals in all three groups receivedintraperitoneal injection of BrdU (50 mg/kg) twice a day for consecutive10 days. After the spinal injury, the animals in the injured/treatmentgroup were administered twice a day via stomach a GASP solution at 8g/kg/day. The injured/control group was administered via stomach a 5%sodium carboxycellulose solution (i.e., the solvent in the GASPsolution). The GASP is provided by Enhan Technology HoldingsInternational Co., Ltd. The thimidine analogue, 5-BrdU, is commonly usedto study DNA synthesis and cell proliferation. It is a nonradioactivemolecule. BrdU is substituted stoichiometrically for thymidine in DNAduring the S-phase (synthesis) of cell growth where the cellular DNAcontent is doubling between the G1 and G2 cell phases. A measure of theincorporated BrdU is related to the new DNA content, i.e., the status orlocation of the cell in the S-phase. Thus, the detection ofBrdU-substituted DNA is functionally related to synthesis of DNA duringthe S-phase of the cell cycle.

Experiment 2 Perfusion, Fixation and Sampling

At 1 day, 2 weeks, and 4 weeks after the spinal injury, the animals inthe corresponding groups were anesthetized by an intraperitonealinjection of 1% pentobarbital sodium. The chest of the anesthetizedanimal was opened and a catheter was inserted from the left ventricle tothe aorta. The animal was first rapidly perfused with physiologicsaline, then followed with perfusion of 4% paraformaldehyde in 0.1 M PBS(pH 7.4) to fix the tissue of the animal. The T8-L1 spinal segment washarvested, placed in a fresh fixation solution and fixed for 4 hours,and then placed in a 30% sucrose solution at 4° C. overnight. The T10,T11, T12, T13 and L1 spinal segments were continuously frozen-sectionedat cross sections to slices of 30 μm thickness.

Experiment 3 Fluorescent Immunohistochemistry

The cross-section slices of spinal cords were first rinsed three timeswith 0.01 M PBS and each time the rinsing lasted for about 5 minutes.The spinal slices were then soaked in 2 N HCl for 30 minutes at 37° C.,followed-by 0.1 M sodium borate buffer (pH 8.3) for 15 minutes and 20.2%Triton X-100 solution at 37° C. for 30 minutes. The spinal slices werethen incubated in normal sheep serum at 37° C. for 20 minutes. MouseantiBrdU primary antibody was added dropwise and the spinal slices werefurther incubated at 37° C. for 2 hours. The spinal slices were rinsedwith 0.01 M PBS three times and each time the rinsing lasted for 5minutes. The biotinylated secondary antibody was then added dropwise andthe spinal slices were incubating at 37° C. for 30 minutes. The spinalslices were rinsed with 0.01 M PBS three times and each time the rinsinglasted for 5 minutes. The SABC-cys3 fluorescent complex solution wasadded dropwise and the spinal slices were incubating at 37° C. for 30minutes. The spinal slices were rinsed with 0.01 M PBS three times whileeach time the rinsing lasted for 5 minutes. For negative control, theprimary antibody was omitted and replaced with PBS. The rest of theprocedures were the same as shown above.

After completing the BrdU staining, the spinal slices of each rat weredivided into 4, each separately treated with the fluorescentimmunohistochemistry stains for nestin (neural stem cell marker), NF(neuron marker), oligodendrocytin (oligodendrocyte marker) and GFAP(astroglial cell marker), respectively.

Under fluorescent microscope at 10×20 magnification, the numbers ofBrdU-positive cells in the ependyma of the central canal of thecross-sectioned spinal slices were counted. For each rat, ten spinalslices were counted to determine the average number of BrdU-positivecells in the ependyma of the central canal per spinal slice. Thecalculated results of each group were expressed as the average numbersof BrdU-positive cells in the ependyma of the central canal±standarddeviation. The t-test on the average numbers of two samples wasperformed.

Results of Examples 1-3:

1. Normal Ependyma in the Spinal Cord Central Canal

In the spinal cross sections of the neck and thoracic regions of thenormal control group, the ependymal cells of the central canal appearedas a round shape, but as an oval shape in the lumbar region. For thepurpose of locating the proliferation of the ependymal cells, mainly theBrdU-positive cells in the ependyma were counted. This was because mostof the BrdU-positive cells were located at the ependyma (FIG. 1). Only afew BrdU-positive cells were distributed in the spinal white matter andgray matter. Detection of nestin expression could lead to thedetermination of the cell counts of the neuron stem cells. The resultsindicated that only a small number of nestin-positive cells in theependyma.

2. Ependymal Cell Proliferation and Nestin Expression in the Spinal CordCentral Canal after Spinal Hemisection

At 1 day after the spinal injury, the ependymal cell layer of thecentral canal was thickened. The BrdU-positive cells markedly increasedafter spinal hemisection (FIG. 2) and more numbers of BrdU-positivecells were observed in the injured animals than in the normal controlanimals. Meanwhile, in the spinal white matter and gray matter, moreBrdU-positive cells were observed after spinal hemisection than thoseseen in the normal control group. This phenomenon was also observed inseveral spinal segments near the injured site. At 2 weeks after thespinal injury, the numbers of BrdU-positive cells in the ependyma wereless than those seen at 1 day after spinal injury but still higher thanthose of the normal control group at 2 weeks. Meanwhile, theBrdU-positive cells of the animals in the injured/treatment group weremore than those of the injured/control group. At 4 weeks after spinalinjury, less BrdU-positive cells, but still significantly higher thanthose of the normal control group, were seen in the ependyma. Thenumbers of BrdU-positive cells of the injured/treatment group werehigher than those of the injured/control group (Table 1). Thus, at 1day, 2 weeks and 4 weeks after spinal injury, there were BrdU-positivecells observed in the ependyma of the spinal cord central canal. As thetime progressed, the number of BrdU-positive cells decreased. TheBrdU-positive cells not only existed in the ependyma of the centralcanal, but also in the spinal white matter and gray matter. The closerto the injured site, the more the BrdU-positive cells in the ependymawere. TABLE 1 Average Numbers of the BrdU-positive cells (x ± s) in theEpendyma of the Spinal Cord Central Canal 4 Weeks After SpinalHemisection average numbers of BrdU- Group numbers of rats positivecells Normal control 2 18.17 ± 2.81 Injured/control 5 29.91 ± 3.68Injured/treatment 5 45.67 ± 3.62t test: pairwise comparison among the 3 groups, P < 0.05

At 1 day after the spinal hemisection, nestin-positive cells in theependyma of the central canal increased. There were also nestin-positivecells in the spinal white matter and gray matter (FIG. 3). At 2 weeksand 4 weeks after spinal injury, there were only a small number ofnestin-positive cells in the ependyma of the central canal while mostdistributed in the spinal white matter and gray matter. In the spinalsegments near the injured site, the white matter ventral to thehemisection injured site had radial nestin-positive protrusions. Alsoseen in other white matter were nestin-positive cell bodies and theirprotrusions (FIG. 4).

At 2 weeks and 4 weeks after the spinal injury, the ependymal cell layerof the spinal central canal of the injured/treatment group wasthickened, having more BrdU-positive cells than those of theinjured/control group. From the results of the BrdU and nestin, BrdU andNF, BrdU and oligodendrocytin, and BrdU and GFAP dualimmunohistochemical stains, it was found that there were no cellspositive with dual stains in the ependyma of the central canal. However,in the spinal white matter of the injured/treatment group there were asmall number of cells positive with dual stains of BrdU and nestin(FIGS. 5A and 5B), BrdU and NF (FIGS. 6A and 6B), BrdU andoligodendrocytin (FIGS. 7A and 7B), and BrdU and GFAP (FIGS. 8A and 8B).

Discussion:

The above study confirmed that at 1 day after the spinal hemisection,the ependymal cells of the central canal proliferated, but the number ofproliferated cells declined as the time progressed. The closer to thespinal injured site, the more the proliferation of the ependymal cellswas. Proliferated cells not only existed in the ependymal cells of thecentral canal, but also in the spinal white matter and gray matter.Meanwhile, some proliferated cells expressed nestin, indicating thesecells had differentiated into neural stem cells. These findings agreedwith the report of Yamamoto et al., Exp. Neurol., 2001; 172(1):115-127,that neural stem cells not only existed in the ependyma of the spinalcentral canal, but also existed in other parts of the spinal cord. Inaddition, the numbers of proliferated ependymal cells of the centralcanal in rats of the injured/treatment group were higher than those ofthe injured/control group, indicating GASP have the effects of promotingcell proliferation of the ependymal cells of the central canal in theinjured spinal cord.

The results of the present study confirmed that after the spinal injury,a small number of proliferated ependymal cells could differentiate intoneural stem cells, neuron-like cells, oligodendrocyte-like cells andastroglial-like cells which possibly involved in the repair of theinjured spinal cord.

CONCLUSION

GASP is effective in promoting the proliferation and/or differentiationof neural stem cells in rats having spinal cord injury.

While the invention has been described by way of examples and in term ofthe preferred embodiments, it is to be understood that the invention isnot limited to the disclosed embodiments. On the contrary, it isintended to cover various modifications as would be apparent to thoseskilled in the art. Therefore, the scope of the appended claims shouldbe accorded the broadest interpretation so as to encompass all suchmodifications.

1. A method for promoting proliferation of neural stem cells in a mammalcomprising: administering an effective amount of sporoderm-brokengermination activated Ganoderma spores (GASP) to a mammal.
 2. The methodaccording to claim 1, wherein said promotion of proliferation of neuralstem cells occur after a spinal cord injury.
 3. The method according toclaim 1, wherein said GASP are orally administered to said mammal. 4.The method according to claim 1, wherein said mammal is human.
 5. Themethod according to claim 1, wherein said spinal cord injury is causedby compression or severance of the spinal cord.
 6. The method accordingto claim 1, wherein said spinal cord injury is caused by a trauma. 7.The method according to claim 1, wherein said spinal cord injury iscaused by a disease.
 8. A method for promoting differentiation of neuralstem cells in a mammal comprising: administering an effective amount ofsporoderm-broken germination activated Ganoderma spores (GASP) to amammal.
 9. The method according to claim 8, wherein said neural stemcells having spinal cord injury.
 10. The method according to claim 8,wherein said promotion of differentiation of said neural stem cellsoccur after a spinal cord injury.
 11. The method according to claim 8,wherein said neural stem cells are differentiated into neurons.
 12. Themethod according to claim 8, wherein said neural stem cells aredifferentiated into astrocytes.
 13. The method according to claim 8,wherein said neural stem cells are differentiated into oligodendrocytes.14. The method according to claim 8, wherein said GASP are orallyadministered to said mammal.
 15. The method according to claim 8,wherein said mammal is human.
 16. The method according to claim 8,wherein said spinal cord injury is caused by compression or severance ofthe spinal cord.
 17. The method according to claim 8, wherein saidspinal cord injury is caused by a trauma.
 18. The method according toclaim 8, wherein said spinal cord injury is caused by a disease.
 19. Amethod for promoting proliferating and differentiating neural stem cellsin a mammal comprising: administering an effective amount ofsporoderm-broken germination activated Ganoderma spores (GASP) to amammal.
 20. The method according to claim 19, wherein said proliferatingand differentiating of said neural stem cells occur after a spinal cordinjury.